PROJECT SUMMARY Salmonella infections are a major health problem worldwide. Salmonella causes disease by expressing genes that are located on pathogenicity islands. Genes that reside on Salmonella Pathogenicity Island-1 (SPI-1) enable Salmonella to adhere to and invade epithelial cells, whereas SPI-2 genes are required for systemic infection. Specialized secretory systems termed type III secretion systems are encoded on each pathogenicity island that provide Salmonella with the means to secrete effector molecules into the host that alter host functions and promote pathogenesis. The present proposal focuses on the control of SPI-2 gene expression. It is one of the most critical virulence determinants of Salmonella, yet the complex molecular biology of its transcriptional regulation, in particular the identification of the pathways for gene expression in vivo, remains poorly defined. Our research is focused on defining these pathways in molecular terms. SPI-2 gene expression is controlled by a two-component regulatory system SsrAB, whose expression is in turn controlled by additional regulatory networks, including the EnvZ-OmpR two-component system, the transcriptional activator SlyA and the global repressor H-NS. The complex regulation of SPI-2 requires integration of multiple environmental signals to ensure that these important virulence genes are expressed at the appropriate time within the macrophage phagosome. In this proposal, critical cis and trans regulatory elements for ssrA/ssrB expression under a variety of environmental conditions will be identified. We hypothesize that OmpR lies at the top of this regulatory hierarchy, activating transcription of the ssrA/B two-component regulatory system. SsrB stimulates expression of the genes encoding the type III secretory apparatus and effectors that are secreted during infection. SsrB is modified by NO stress during macrophage infection, the consequences of this cysteine-modification to expression of SPI-2 genes will be examined in both mouse macrophages, a macrophage-like cell line and mouse tissues upon infection with Salmonella wild type and ssrB mutant strains. As a result of our studies, we will have an enhanced understanding of the molecular events that occur as a result of Salmonella infection and how these modifications alter gene expression in the host. PUBLIC HEALTH RELEVANCE: Impact on Veteran's Health Veterans suffer from both acute and chronic bacterial infections. Our studies are mechanistic and have implications beyond Salmonella, extending to other pathogens. OmpR has been shown to be required for virulence in Vibrio cholerae, Shigella, Yersinia and other infectious species. Furthermore, drug-resistant Salmonella infections are a problem in all hospitals, including VA hospitals and septicemia due to Salmonella is a problem in immune-compromised HIV positive patients, a high incidence which occurs in veterans. Salmonella-associated acute gastroenteritis is a significant cause of morbidity among travelers, deployed military personnel and high population density closed communities (e.g. military bases, hospitals or nursing homes). The number of typhoid fever cases exceeds 33 million per year worldwide and remains a potential concern among previously vaccinated travelers and military personnel.